Diagnosis of anaplastic lymphoma kinase rearrangement in cytological samples through a fluorescence in situ hybridization–based assay: Cytological smears versus cell blocks

Abstract

Anaplastic lymphoma kinase (ALK) status analysis of lung cytological specimens should be successfully encouraged in routine practice because biopsy specimens are not always available. To date, the US Food and Drug Administration has approved both fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) as diagnostic tests for identifying ALK-positive patients eligible for treatment with crizotinib. Although ALK IHC is an optimal diagnostic tool, FISH becomes mandatory in equivocal cases. ALK FISH of paraffin-embedded tissue material is still the gold standard, whereas the cytological specimen assay has not yet been completely standardized. Many controversial data have been reported on the adequacy of cytology cell blocks (CBs) versus conventional smears for FISH testing. This review discusses some critical issues related to ALK FISH of cytological samples, including the triaging of collected specimens to optimize the material, the use of CBs versus conventional smears, and alternative methods for an ALK rearrangement diagnosis. Conventional smears have the advantages of an immediate evaluation, no probe tissue-related artifactual loss, no fixation-related alterations, and usually sufficient material for an analytic preparation. On the other hand, CBs have several advantages, including the appropriate conservation of the tissue architecture, an absence of problems related to cell overlapping, and the ability to evaluate neoplastic cells in a dark field. Cancer Cytopathol 2017;125:303-312. © 2017 American Cancer Society.

ALK and ROS1 testing on lung cancer cytologic samples: Perspectives

“…52 Indeed, recent evidence indicates that ALK FISH testing using direct smears, which circumvents nuclear truncation and ensures better DNA preservation than FFPE samples, is highly sensitive. 53 In a cytology study of 32 lung adenocarcinomas analyzed for ALK rearrangements using matched FISH and smears, concordance was obtained in 31 of 32 cases (97%). In the 1 discordant case, an ALK rearrangement was detected on the direct smear, and RT-PCR confirmed the rearrangement.…”

“…Although the use of cell block sections for ALK testing is still recommended over smears, a draft document circulated for open comment preliminary to the upcoming College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology revised recommendations acknowledges the use of conventional cytologic preparations for ALK FISH . Indeed, recent evidence indicates that ALK FISH testing using direct smears, which circumvents nuclear truncation and ensures better DNA preservation than FFPE samples, is highly sensitive . In a cytology study of 32 lung adenocarcinomas analyzed for ALK rearrangements using matched FISH and smears, concordance was obtained in 31 of 32 cases (97%).…”

“…Thus, ThinPrep‐FISH can be used for testing when the cell block is paucicellular. The preanalytic steps, ie, before hybridization, affect the interpretation of probe signals . In a recent study of 91 smears, mostly represented by Cytofix‐processed (BD Biosciences, Franklin Lakes, New Jersey) and Papanicolaou‐stained slides, 24 (26%) could not be evaluated because of artifacts or cell overlap.…”

“…The preanalytic steps, ie, before hybridization, affect the interpretation of probe signals. 53 In a recent study of 91 smears, mostly represented by Cytofix-processed (BD Biosciences, Franklin Lakes, New Jersey) and Papanicolaou-stained slides, 24 (26%) could not be evaluated because of artifacts or cell overlap. FISH also failed in 3 of 17 cases(17%) because of cell detachment when carried out on the Diff-Quikstained smear used for ROSE.…”

“…55 Moreover, the performance of FISH depends on several preanalytic factors, namely, fixation time and fixative type. 53,63 Consequently, current expert opinion holds that an ALKpositive cell rate close to 15% is "borderline" and requires additional evaluation. 64…”